A number of Gram-negative pathogens actively secrete enzymes and toxins into their surrounding medium. This process is controlled in Pseudomonas aeruginosa by a complex machinery consisting of at least 12 proteins. This extracellular secretion machinery shares similarities with type IV pili biogenesis. Precursors of type IV pilins and four of the components of the extracellular secretion machinery are homologues and they are all post- translationally processed by enzyme PilD, a bifunctional leader peptidase/N-methylase. Other genes encoding determinants of pilus biogenesis and extracellular protein secretion also share sequence similarity. This proposal will examine the composition and function of the protein secretion machinery of P. aeruginosa. The structure-function analysis of PilD will be the initial focus of research. Using genetic and biochemical techniques, the active domains of this bifunctional enzyme will be mapped. Amino acids that make up the protease and methylase sites will be identified by suppressor analysis of mutants in PilD-substrate interactions. Isolation of mutants lacking methyl-transferase activity will allow a more careful assessment of the importance of methylation in extracellular protein secretion and pilus formation. One of the components of the extracellular secretion machinery is XcpR, a protein containing a putative ATP-binding site. The interaction of this protein with other components of the export apparatus in vivo and in vitro will be studied. Attempts will be made to determine whether XcpR hydrolyzes ATP as part of its function during protein secretion. The ATP binding region will be defined by crosslinking of XcpR with ATP. The existence of an assembled organelle responsible for protein secretion will be probed by chemical crosslinking and co-immunopreciptiation. Using antibodies to the various components of the secretory apparatus, interactions between proteins of the export machinery in the bacterial cell envelope will be investigated. The genetic organization of the genes specifying export determinants will be studied in order to determine the regulatory mechanisms that control their coordinate expression necessary for assembly of a functional secretory apparatus. It is hoped that the results of this work will provide insight into basic mechanisms of extracellular protein secretion. Insights gained from research in P. aeruginosa may be applicable to a number of different pathogens that export proteins by a similar mechanism.